Human beings suffer serious health risks when exposed to certain chemical substances. Among these risks is the effect of genetic alteration caused by chemically-induced mutation. The results of such genetic alteration can be devastating, as for example, when the mutation is to the DNA of a gamete contributing critical genetic information to a developing embryo. Also, chemically-induced mutation in somatic cells may be a contributing cause to many cancers.
Investigators have developed mutagenicity assays that measure the ability of chemical agents or physical agents such as an X-ray to cause an alteration in the DNA of an organism. Such assays may be used to predict the carcinogenic potential of the agent on the basis of its ability to induce mutations in cultured cells.
Mutagenicity assays typically involve exposing a cell culture to an agent. After exposure is terminated, the culture is allowed to grow for a period of time necessary to allow a mutant phenotype to be expressed. The frequency of the mutant phenotype in the test culture then is compared to a control culture to determine whether exposure induced a higher frequency of mutation.
A useful characteristic which may be used to determine the occurrence of a mutation is a cell's ability to make an enzyme after exposure to a potential mutagen. A cell may possess an enzyme that will convert a non-toxic substance into a toxin. Such cells will die when grown in the presence of the non-toxic precursor. A mutation to a gene related to the enzyme may eliminate the enzyme as a product of the cell. Such a mutated cell not producing the enzyme will live when grown in the presence of the non-toxic precursor because it will not have the ability to convert the non-toxic precursor into a toxin. Thus, the existence of the mutation may be determined by the ability of a cell to grow in media containing the non-toxic precursor.
Mutation assays have been performed using a number of precursors of this nature, including the following: 6-thioguanine (6-TG), which kills cells that contain hypoxanthine-guanine-phosphoriboxyl transferase (HGPRT)[1]; 8-azaguanine, which kills cells that contain HGRPT [2]; and trifluorodeoxythymidine, which kills cells that contain thymidine kinase [3].
Cells from various organisms have been used in mutagenicity assays, including cells from bacteria [4, 5] yeast [6], hamsters [7, 8], mice [9] and humans [10, 11, 12, 13]. However, species differ in their sensitivity to various substances. Thus, data from assay systems incorporating human cells should be most predictive of potential effects in human populations.
A considerable body of evidence indicates that it is the metabolites of many xenobiotics which are ultimately responsible for the mutagenic effect rather than the xenobiotic compounds themselves. However, cell lines grown in long-term cell culture tend to lose their ability to metabolize xenobiotics and therefore cannot be used to accurately predict the potential carcinogenicity of a substance. To overcome this problem, an exogenous source of xenobiotic metabolism has been provided in various mutagenicity assays.
Two general approaches have been taken to provide xenobiotic metabolism in in vitro mutagenicity assays. The most common approach is to use a homogenate of a tissue rich in the enzymes responsible for the metabolism of xenobiotics, usually rat liver [14, 15]. However, such exogenous homogenates are unsatisfactory because they come from rats, not humans, and because they are known to metabolize chemicals differently than the intact tissue [16]. Furthermore, the use of exogenous homogenates complicates the procedure by requiring additional steps, including careful control of pH and sterility. Moreover, the addition of such homogenates creates conditions different from those existing normally in vivo.
Another approach is to co-cultivate metabolically-competent cells with the suitable target cells [17, 18]. This approach, however, requires unstable, chemically-reactive metabolites to traverse from the metabolically-competent cells to the DNA of the target cells, with a concomitant loss of sensitivity.
One recent improvement to the foregoing mutagenicity assay systems is described in U.S. Pat. No. 4,532,204, the disclosure of which is incorporated by reference. This patent describes a newly-isolated and purified human cell line, designated as AHH-1 cells. The AHH-1 cell line is described as being isolated from a culture of lymphoblast cells supplied by the Roswell Park Memorical Institute (RPMI-1788 cells). The cell line was selected to have: rapid growth rate and high cloning efficiency; the absence of mycoplasma; the ability to grow in suspension; and a stable near diploid genome. In addition, the AHH-1 cell line was selected to have high levels of oxidative activity, as compared to the RPMI-1788 cells and in particular high aryl hydrocarbon hydroxylase (AHH) activity. This activity is due primarily to the cytochrome P450IA1 (for the purposes of this document we employ the Cytochrome P450 nomenclature described in Nebert et al, 1987 DNA 6:1-11) mono-oxygenase responsible for converting certain xenobiotic compounds into mutagenic metabolites. Thus, it was stated that the AHH-1 cell line does not require an exogenous source of cytochrome P450IA1 for mutagenicity studies.
At least four characteristics of AHH-1 cells limit their usefulness in mutagenicity assays and metabolic transformation studies:
(1) The induction of gene mutations can only be measured at the hemi-zygous (X-linked) HGPRT locus; however, recovery of induced mutants may be much higher at autosomal loci. PA1 (2) The level of cytochrome P450IA1 activity in AHH-1 cells, while sufficient to activate some chemicals to their mutagenic form, is unacceptably low for other chemicals. Therefore, the mutagenicity of certain chemicals inefficiently acted on by cytochrome P450IA1 will go undetected in a mutagenicity assay using AHH-1 cells. PA1 (3) AHH-1 cells do not detectably express cytochrome P450 mono-oxygenase of the P450II class. Members of this class convert certain substances such as nitrosamines, into mutagenic metabolites which are of considerable concern to human health. AHH-1 cells are relatively insensitive to the mutagenic effects of nitrosamines and, therefore, AHH-1 cells will not detect the mutagenic effect of this important class of compounds. PA1 (4) AHH-1 cells do not express detectable levels of microsomal epoxide hydrolase (E.C. 3.3.2.3) and thus do not produce the known mutagenic metabolites of polycyclic aromatic hydrocarbons that are found in vivo.
The AHH-1 cell line does not have the oxidative activity found in vivo and responsible for the metabolism of considerably large classes of xenobiotics into mutagenic end products. A human cell line stably expressing the genes responsible for this oxidative activity would be very desirable. One group recently has reported the transfer of mouse cytochrome P-450 genes into mouse and human cells using a viral vector [19]. These genes were expressed in both populations of cells infected with the recombinant virus for up to ten hours after infection. Mutagenicity and other toxicological assays, however, require stable and homogenous expression of metabolizing enzymes which was not demonstrated in that study.
The cytochromes P450 are a large family of hemoproteins capable of metabolizing xenobiotics such as drugs, procarcinogens and environmental pollutants as well as endobiotics such as steroids, fatty acids and prostaglandins. Many mammalian cell lines have lost most or all capacity to perform cytochrome P450-catalyzed reactions. This limitation has restricted studies of cytochrome P450 mediated metabolism to either primary cells or tissue homogenates. The lack of adequate endogenous cytochrome P450's has also led to the incorporation of extracellular metabolizing systems, commonly rodent liver homogenates, into assays designed to detect genotoxic effects of promutagens and procarcinogens.
Recently, several laboratories have successfully transfected cytochrome P450 cDNAs into mammalian cell lines. Virtually all of the cell lines were developed by transfecting a single P450 cDNA into the target cell line. While these cell lines may be quite useful for examining P450 specific activation of procarcinogens, given the multiplicity of P450 forms expressed in vivo, it is clear that the use of cell lines expressing single P450s to screen compounds for mutagenic activity will be a daunting task because of the number of cell lines needed. Accordingly, it would be desirable to utilize a small number of cell lines expressing a multiplicity of P450s. It would be even more desirable to have and utilize a single cell line stably expressing the all of the human P450 forms primarily responsible for the activation of procarcinogens in human microsomes.
The task of creating a cell line stably expressing a multiplicity of P450s has clear obstacles. Because the P450 genes have substantial homology, it would be expected that a single cell line transfected with multiple P450s would be unstable due to the likelihood of inter or intra vector recombination. Additionally, instability further would be expected in a cell-line containing multiple transfected promoters due to the promoters interfering with one another. These and other obstacles are overcome by the methods and cell lines of the invention.